Process for solubilizing glucagon-like peptide 1 compounds

ABSTRACT

Disclosed is a method of preparing a GLP-1 compound that is soluble in aqueous solution at pH 7.4 from a GLP-1 compound that is substantially insoluble in aqueous solution at pH 7.4. The insoluble GLP-1 compound is dissolved in aqueous base or in aqueous acid to form a GLP-1 solution. The GLP-1 solution is then neutralized to a pH at which substantially no amino acid racemization of the GLP-1 compounds occurs, after which the soluble GLP-1 compound is isolated from the neutralized solution.

This application is a continuation of U.S. application Ser. No.12/551,841, filed Sep. 01, 2009, which is a continuation of U.S.application Ser. No. 10/169,657, now U.S. Pat. No. 7,598,222, filed Jan.16, 2001, which claims the priority of U.S. Provisional Application Nos.60/178,438, filed Jan. 27, 2000 and 60/224,058, filed Aug. 09, 2000.

Glucagon-Like Peptide 1 (GLP-1) is a 37 amino acid peptide that issecreted by the L-cells of the intestine in response to food ingestion.It has been found to stimulate insulin secretion (insulinotropicaction), thereby causing glucose uptake by cells and decreased serumglucose levels (see, e.g., Mojsov, S., Int. J. Peptide Protein Research,40:333-343 (1992)). However, GLP-1 (1-37) is poorly active. A subsequentendogenous cleavage between the 6^(th) and 7^(th) position produces amore potent biologically active GLP-1 (7-37)OH peptide. Numerousbiologically active GLP-1 analogs and derivatives, referred to herein as“GLP-1 compounds” are also known. For example, GLP-1 (7-36)NH₂ is anaturally occurring GLP-1 analog in which glycine at the C-terminal hasbeen replaced with —NH₂. Val⁸-GLP-1 (7-37)OH is a synthetic GLP-1(7-37)OH analog in which alanine at position 8 has been replaced withvaline; and Thr¹⁶-Lys¹⁸-GLP-1 (7-37)OH is a synthetic GLP-1 (7-37)OHanalog in which valine at position sixteen and serine at positioneighteen have been replaced with threonine and lysine, respectively.Because of their ability to stimulate insulin secretion, GLP compoundsshow great promise as agents for the treatment of diabetes, obesity, andrelated conditions.

GL-1 compounds can exist in at least two different forms. The first formis physiologically active and dissolves readily in aqueous solution atphysiological pH (7.4). In contrast, the second form has little or noinsulinotropic activity and is substantially insoluble in water at pH7.4. Unfortunately, the inactive form is readily produced when aqueousGLP-1 solutions are agitated, exposed to hydrophobic surfaces or havelarge air/water interfaces. This considerably complicates the productionof commercial quantities of active GLP-1 compounds; mixing operations orcontinuous movement through a pump are common operations in bulkmanufacturing processes and these operations cause the agitation,air/water interfaces and/or contact with hydrophobic surfaces thatresults in the insoluble form.

The realization of the pharmaceutical potential of GLP-1 compounds isdependent on the production of the active form of GLP-1 compounds incommercially viable quantities without contamination with significantquantities of by-products of the inactive form. Thus, there is acritical need for methods of converting in high yield bulk amounts ofthe inactive insoluble form of GLP-1 compounds to the soluble activeform.

It has now been found that the inactive, insoluble form of GLP-1compounds can be converted into the physiologically active, soluble formby dissolving the inactive form in aqueous base (or in aqueous acid). Afurther discovery, reported herein, is that the soluble, physiologicallyactive form of GLP-1 compounds can be isolated in high yield and withoutamino acid racemization or other degradation, provided that the aqueousbase solution (or aqueous acid solution) is neutralized to a suitable,less basic (or less acidic) pH. For example, insoluble Val⁸-GLP-1(7-37)OH was converted to soluble Val⁸-GLP-1 (7-37)OH in high yield andwithout detectable racemization by dissolving in aqueous sodiumhydroxide at pH 12.3, neutralizing to pH 7.0 and isolating the solubleproduct by filtration and lyophilization (Examples 4 and 6). Based onthese discoveries, a method of preparing a soluble, physiologicallyactive GLP-1 compound from its corresponding inactive, insoluble form isdisclosed.

The present invention is a method of preparing a GLP-1 compound that issoluble in aqueous solution at pH 7.4 from a GLP-1 compound that issubstantially insoluble in aqueous solution at pH 7.4. The insolubleGLP-1 compound is dissolved in aqueous base or in aqueous acid to form aGLP-1 solution. The GLP-1 solution is then neutralized to a pH at whichsubstantially no amino acid racemization of the GLP-1 compounds occurs.The soluble GLP-1 compound is then isolated from the neutralizedsolution.

In another embodiment, the present invention is a method of convertingan insoluble GLP-1 compound in a composition comprising said insolubleGLP-1 compound to a soluble GLP-1 compound. The composition comprisingthe insoluble GLP-1 compound is dissolved in aqueous acid or in aqueousbase to form a GLP-1 solution. The GLP-1 solution is then neutralized toa pH at which substantially no amino acid racemization of the GLP-1compounds occurs. A composition comprising soluble GLP-1 compound isthen isolated from the neutralized solution.

The method of the present invention can be used to convert bulkquantities of the inactive form of GLP-1 compounds to the active formwithout significant amino acid racemization or other degradation.Therefore, it can be used in commercial processes to prepare activeGLP-1 compounds in high yield and in high purity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the conversion over time of the soluble formof three different lots of Val⁸-GLP(7-37)OH to the insoluble form insolution (pH 7.4, no salt, 20 mM phosphate, 1 mg/mL protein) withstirring (labeled “s”) and without stirring.

A GLP-1 compound is a peptide having from about twenty-five to aboutthirty-five naturally occurring or modified amino acids and hassufficient homology to GLP-1 (7-37)OH such that it exhibitsinsulinotropic activity. A “modified amino acid” is the amino acidobtained after one or more chemical modifications to a naturallyoccurring amino acid. Examples of modified amino acids are providedbelow in the definition of “GLP-1 derivative”. “Insulinotropic activity”refers to stimulating insulin secretion in response to food ingestion,thereby causing glucose uptake by cells and decreased serum glucoselevels. A wide variety of GLP-1 compounds are known in the art,including GLP analogs, GLP derivatives, biosynthetic GLPs anddipeptidyl-peptidase-IV protected GLPs. The meaning of the terms “GLPanalog”, “GLP derivative”, “biosynthetic GLP” and“dipeptidyl-peptidase-IV protected GLP” are provided herein below.

The solubility of a GLP-1 compound can vary, depending upon how it hasbeen isolated and the manner and length of time it has been stored. Forexample, the solubility of GLP-1 compounds which have been isolated fromsolution by crystallization or lyophilization is generally very high. Incontrast, the solubility of a GLP-1 compound which precipitates from asolution that contains high salt concentrations, has been exposed tohydrophobic surfaces (e.g., purified by tangential flow filtration) orhas large air/water interfaces resulting from, for example, vigorousstirring, is often very low (Example 2). In addition, highly solublecrystals and lypholized powders of a GLP-1 compound typically showdecreased solubility over time. Storage of the compound at lowtemperatures (e.g., at 0° C.) can slow the conversion to less solubleforms.

The degree to which a GLP-1 compound is soluble in water atphysiological pH can be correlated to the insulinotropic activity of thecompound. Specifically, the biological activity of a GLP-1 compoundincreases as it becomes more soluble and decreases as it becomes lesssoluble. Insulinotropic activity can be assessed by methods known in theart, including using in vivo experiments such as is described in Example5 and in vitro assays employing pancreatic islet cells or insulinomacells, as described in EP 619,322 to Gelfand, et al., and U.S. Pat. No.5,120,712, respectively. The entire teachings of these references areincorporated herein by reference.

The degree to which a GLP-1 compound is soluble in water atphysiological pH can also be correlated to absorbances in the infraredspectrum. Specifically, the infrared spectrum of GLP-1 compounds ischaracterized by absorbances at 1624 cm⁻¹, 1657 cm⁻¹ and 1696 cm⁻¹. Thesolubility of the GLP-1 compound increases with the intensity of theabsorbance at 1657 cm⁻¹ and decreases with the intensity of theabsorbances at 1624 cm⁻¹ and 1696 cm⁻¹. Thus, the intensity of thesethree absorbances will change accordingly as a GLP-1 compound convertsto a more soluble or less soluble form (see Example 3).

A GLP-1 compound having a solubility in water at physiological pH (7.4)of at least about 1.0 milligrams per milliliter of water at pH 7.4 issaid to be in the “active form” or “soluble form” of the compound. AGLP-1 compound in its active or soluble form is referred to herein as a“soluble GLP-1 compound”. Preferably, a soluble GLP-1 compound has asolubility in water at physiological pH of at least about 5.0 milligramsper milliliter. The ratio A₁₆₅₇/(A₁₆₂₄+A₁₆₅₇+A₁₆₉₆) is generally atleast about 0.60 for GLP-1 compounds in the soluble form and preferablyat least about 0.70. A_(n) is the absorbance intensity at wavelength nin recipricol centimeters.

A GLP-1 compound having a solubility in water at physiological pH lessthan about 0.5 milligrams per milliliter of water is said to be in the“inactive form” or “insoluble form” of the compound. A GLP-1 compound inits inactive or insoluble form is referred to herein as an “insolubleGLP-1 compound”. Preferably, an insoluble GLP-1 compound has asolubility in water at physiological pH of less than about 0.1milligrams per milliliter. The ratio (A₁₆₂₄+A₁₆₉₆)/(A₁₆₂₄+A₁₆₅₇+A₁₆₉₆)is generally at least about 0.60 for GLP-1 compounds in the soluble formand preferably at least about 0.70. A_(n) is the absorbance intensity atwavelength n in recipricol centimeters.

It has been reported that the insoluble form of GLP-1 compounds ischaracterized by the formation of intramolecular and intermolecular betasheets, which results in compound aggregation and insolubility, whereasthe secondary structure of the soluble form is characterized by thepresence of alpha helices (see Senderoff et. al. J. Pharm. Sci. 87:183(1998), the entire teachings of which are incorporated herein byreference). The infrared spectrum of the soluble and insoluble forms ofGLP-1 compounds is consistent with this interpretation. Specifially,absorbance bands at 1624 and 1696 cm⁻¹ are generally indicative of thepresence of beta sheets, whereas an absorbance band at 1657 cm⁻¹ isconsistent with the presence of alpha helices.

The dissolution of insoluble GLP-1 compounds in aqueous base (or aqueousacid) is consistent with the breakdown of the intramolecular andintermolecular interactions responsible for beta sheet formation.Moreover, the isolation of the soluble form of GLP-1 compounds fromthese solutions is consistent with the reformation of the secondarystructure of soluble GLP-1 compounds. Therefore, the method of thepresent invention can be used with proteins (e.g., GLP-1 compounds)having a soluble active form characterized by high alpha helix contentand an inactive or insoluble form characterized by high beta sheetcontent to convert from the insoluble to the soluble form.

The method of the present invention can be used to convert insolubleGLP-1 compounds to soluble GLP-1 compounds in compositions wherein theinsoluble GLP-1 compound is the only component or, alternatively,wherein the insoluble GLP-1 compound is one of several components. Thus,the method can “purify” mixtures comprising both the soluble andinsoluble form of a GLP-1 compound by converting the insoluble form tothe soluble form. The method is therefore ideally suited for removingsmall or even trace amounts of the insoluble form from or minimizing theamount of the insoluble form in the composition before, for example,administration as a drug or formulation into a drug dosage form.

By custom in the art, the amino terminus of GLP-1 (7-37)OH has beenassigned number residue 7 and the carboxy-terminus, number 37. Thisnomenclature carries over to other GLP compounds. When not specified,the C-terminal is usually considered to be in the traditional carboxylform. The amino acid sequence of GLP-1 (7-37)OH is provided below:

(SEQ ID NO: 1) ⁷His-Ala-Glu-¹⁰Gly-Thr-Phe-Thr-Ser-¹⁵Asp-Val-Ser-Ser-Tyr-²⁰Leu-Glu-Gly-Gln-Ala-²⁵Ala-Lys-Glu-Phe-Ile-³⁰Ala-Trp-Leu-Val-Lys-³⁵Gly-Arg-³⁷Gly-COOH

A “GLP-1 compound” has sufficient homology to GLP-1 (7-37)OH or afragment of GLP-1 (7-37)OH such that the compound has insulinotropicactivity. Preferably, a GLP-1 compound has the amino acid sequence ofGLP-1 (7-37)OH or a fragment thereof, modified so that from zero, one,two, three, four or five amino acids differ from the amino acid incorresponding position of GLP-1 (7-37)OH or the fragment of GLP-1(7-37)OH. Preferred GLP compounds are modified at position 8, atposition 22 or at position 8 and position 22.

Preferably, the amino acids in the GLP compound which differ from theamino acid in corresponding position of GLP-1(7-37)OH are conservativesubstitutions and, more preferably, are highly conservativesubstitutions.

A “conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in the their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Listed below are five groups of amino acids. Replacing an amino acid ina GLP-1 compound with another amino acid from the same groups results ina conservative substitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, methionine and non-naturally occurring        amino acids with C1-C4 aliphatic or C1-C4 hydroxyl substituted        aliphatic side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, ornithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C1-C4 aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

A “highly conservative substitution” is the replacement of an amino acidwith another amino acid that has the same functional group in the sidechain and nearly the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have nearly the same sizewhen the total number carbon and heteroatoms in their side chainsdiffers by no more than two. They have nearly the same shape when theyhave the same number of branches in the their side chains. Example ofhighly conservative substitutions include valine for leucine, threoninefor serine, aspartic acid for glutamic acid and phenylglycine forphenylalanine. Examples of substitutions which are not highlyconservative include alanine for valine, alanine for serine and asparticacid for serine.

“GLP-1 analog” is defined as a GLP-1 compound having one or more aminoacid substitutions, deletions, inversions, or additions relative toGLP-1 (7-37)OH. Permissible amino acid substitutions include thereplacing of an L-amino acid with its corresponding D-form. In thenonmenclature used herein to designate GLP-1 analogs, the substitutingamino acid and its position is indicated prior to the parent structure.For example, Val⁸-GLP-1 (7-37)OH designates a GLP-1 analog in which thealanine normally found at position eight of GLP-1 (7-37)OH has beenreplaced with valine. Numerous GLP-1 analogs are known in the art andinclude, but are not limited to: GLP-1 (7-34), GLP-1 (7-35), GLP-1(7-36)NH₂, Gln⁹-GLP-1 (7-37), d-Gln⁹-GLP-1 (7-37),Thr¹⁶-Lys¹⁸-GLP-1(7-37), Lys¹⁸-GLP-1 (7-37), Gly⁸-GLP-1 (7-36)NH₂,Gly⁸-GLP-1 (7-37)OH, Val⁸-GLP-1 (7-36)NH₂, Val⁸-GLP-1 (7-37)OH,Met⁸-GLP-1 (7-36)NH₂, Met⁸-GLP-1 (7-37)OH, Ile⁸-GLP-1 (7-36)NH₂,Ile⁸-GLP-1 (7-37)OH, Thr⁸-GLP-1 (7-36)NH₂, Thr⁸-GLP-1(7-37)OH,Ser⁸-GLP-1 (7-36)NH₂, Serb-GLP-1 (7-37)OH, Asp⁸-GLP-1(7-36)NH₂,Asp⁸-GLP-1 (7-37)OH, Cys⁸-GLP-1 (7-36)NH₂, Cys⁸-GLP-1 (7-37)OH,Thr⁹-GLP-1 (7-37), D-Thr⁹-GLP-1 (7-37), Asn⁹-GLP-1 (7-37), D-Asn⁹-GLP-1(7-37), Ser²²-Arg²³-Arg²⁴-Gln²⁶-GLP-1 (7-37), Arg²³-GLP-1 (7-37),Arg²⁴-GLP-1 (7-37), and Gly⁸-Gln²¹-GLP-1 (7-37)OH, and the like.

A “GLP-1 derivative” is defined as a molecule having the amino acidsequence of GLP-1 (7-37) or of a GLP-1 analog, but additionally havingchemical modification of one or more of its amino acid side groups,α-carbon atoms, terminal amino group, or terminal carboxylic acid group.A chemical modification includes, but is not limited to, adding chemicalmoieties, creating new bonds, and removing chemical moieties.Modifications at amino acid side groups include, without limitation,acylation of lysine ε-amino groups, N-alkylation of arginine, histidine,or lysine, alkylation of glutamic or aspartic carboxylic acid groups,and deamidation of glutamine or asparagine. Modifications of theterminal amino group include, without limitation, the des-amino, N-loweralkyl, N-di-lower alkyl, and N-acyl (e.g., —CO-lower alkyl)modifications. Modifications of the terminal carboxy group include,without limitation, the amide, lower alkyl amide, dialkyl amide, andlower alkyl ester modifications. Lower alkyl includes straight orbranched chain C₁-C₄ alkyl. Furthermore, one or more side groups, orterminal groups, may be protected by protective groups known to theordinarily-skilled protein chemist. The α-carbon of an amino acid may bemono- or dimethylated.

Other GLP-1 compounds are described in U.S. Pat. No. 5,705,483 and havethe amino acid sequence shown by SEQ ID NO: 2. The entire teachings ofU.S. Pat. No. 5,705,483 are incorporated herein by reference.

(SEQ ID NO: 2) R₁-X-Glu-¹⁰Gly-Thr-Phe-Thr-Ser-¹⁵Asp-Val-Ser-Ser-Tyr-²⁰Leu-Y-Gly-Gln-Ala-²⁵Ala-Lys-Z-Phe-Ile-³⁰Ala-Trp-Leu-Val-Lys-³⁵Gly-Arg-R₂

R₁ in SEQ ID NO: 2 is L-histidine, D-histidine, desamino-histidine,2-amino-histidine, beta-hydroxy-histidine, homohistidine,alpha-fluoromethyl-histidine or alpha-methyl-histidine.

X is Ala, Gly, Val, Thr, Met, Ile, Ser or alpha-methyl-Ala.

Y is Glu, Gln, Ala, Thr, Ser or Gly.

Z is Glu, Gln, Ala, Thr, Ser or Gly.

R₂ is NH₂ or Gly-OH.

Yet other GLP-1 compounds are described in WO 91/11457, the entireteachings of which are incorporated herein by reference. These GLP-1compounds include GLP-1 (7-34), GLP-1(7-35), GLP-1 (7-36), or GLP-1(7-37), or the amide form thereof, and pharmaceutically-acceptable saltsthereof, having at least one of the following modifications:

(a) substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,phenylalanine, arginine, or D-lysine for lysine at position 26 and/orposition 34; or substitution of glycine, serine, cysteine, threonine,asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine,methionine, phenylalanine, lysine, or a D-arginine for arginine atposition 36;

(b) substitution of an oxidation-resistant amino acid for tryptophan atposition 31;

(c) substitution of at least one of: tyrosine for valine at position 16;lysine for serine at position 18; aspartic acid for glutamic acid atposition 21; serine for glycine at position 22; arginine for glutamineat position 23; arginine for alanine at position 24; and glutamine forlysine at position 26;

(d) substitution of at least one of: glycine, serine, or cysteine foralanine at position 8; aspartic acid, glycine, serine, cysteine,threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,leucine, methionine, or phenylalanine for glutamic acid at position 9;serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine,valine, isoleucine, leucine, methionine, or phenylalanine for glycine atposition 10; and glutamic acid for aspartic acid at position 15; or

(e) substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,or phenylalanine, or the D- or N-acylated or alkylated form of histidinefor histidine at position 7; wherein, in the substitutions in (a), (b),(d), and (e), the substituted amino acids can optionally be in theD-form and the amino acids substituted at position 7 can optionally bein the N-acylated or N-alkylated form.

Yet other GLP-1 compounds are disclosed in U.S. Pat. No. 5,188,666, theentire teachings of which are incorporated herein by reference. Examplesinclude a peptide having the amino acid sequence of SEQ ID NO: 3:

(SEQ ID NO: 3) His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Xand pharmaceutically-acceptable salts thereof.

X in SEQ ID NO: 3 is Lys-COOH and Lys-Gly-COOH.

Also included are a pharmaceutically-acceptable lower alkyl ester ofsaid peptide and a pharmaceutically-acceptable amide of said peptide,e.g., lower alkyl amide, and lower dialkyl amide. Generally, lower alkylgroups are a C1-C20 straight chained or branched aliphatic group withzero, one or more units of unsaturation.

Yet other GLP-1 compounds are disclosed in U.S. Pat. No. 5,512,549, theentire teachings of which are incorporated herein by reference. TheseGLP-1 compounds have the amino acid sequence of SEQ ID NO: 4:

R¹ is 4-imidazopropionyl, 4-imidazoacetyl, or 4-imidazo-α, αdimethyl-acetyl.

R² is C₆-C₁₀ unbranched acyl or is absent.

R³ is Gly-OH or NH₂.

Xaa is Lys or Arg.

Still other GLP-1 compounds are disclosed in WO 98/08871, the entireteachings of which are incorporated herein by reference. These GLP-1compounds include a lipophilic substituent attached to the N-terminal orto the C-terminal amino acid residue wherein the substituent is an alkylgroup or a group which has an omega carboxylic group.

“Biosynthetic GLP-1's” are defined as any GLP-1 analog or nativesequence which contain only naturally occurring amino acid residues andare thus capable of being expressed by living cells, includingrecombinant cells and organisms.

“DPP-IV protected GLP's” refers to GLP-1 compounds which are resistantto the action of DPP-IV. These include analogs having a modified or Damino acid residue in position 8. These also include biosyntheticGLP-1's having Gly or the L amino acid residues Val, Thr, Met, Ser, Cys,Ile, or Asp in position 8. Other DPP-IV protected GLP-1 compoundsinclude des amino His⁷ and other His⁷ derivatives.

In the method of the present invention, insoluble GLP-1 compound isadded to a solution that is sufficiently basic (or acidic) to dissolvethe compound. The solution is then mixed or otherwise agitated until thecompound dissolves. GLP-1 compounds generally dissolve more rapidly asthe basicity or acidity of the solution increases. However, the rate ofracemization and other degradation also increases as the solutionbecomes more basic (or acidic) and the longer a GLP-1 compound isdissolved in highly basic or acidic solution. Therefore, in practicingthe method of the present invention, it is desirable to use solutionsthat are sufficiently basic (or acidic) to readily dissolve the compoundbut not so basic (or acidic) to result in amino acid racemization or theformation of other by-products. The skilled artisan will be able toroutinely identify suitable pHs which will achieve these dual goals.Basic solutions having a pH between about 10.5 and about 12.5 and acidicsolutions having a pH between about 1.5 and about 2.0 are typically usedfor carrying out the method of the present invention. However, lessbasic solutions can also be used, provided that sufficient period oftime is allowed for dissolution. Solutions having a pH between about10.0 and 10.5 have been used with dissolution times of at least thirtyminutes. Preferred are pHs between about 12.1 and about 12.5. In oneaspect, it is desirable to minimize the amount of aqueous base oraqueous acid being used to dissolve the GLP-1 compound. A minimum amountof aqueous base or aqueous acid is preferred when the final GLP-1compound is being isolated by lyophilization.

Following dissolution of the GLP-1 compound, the solution, referred toherein as a “GLP-1 solution”, is neutralized to a pH at whichsubstantially no amino acid racemization occurs, e.g., less than 1% ofthe GLP-1 compound racemizes per hour and preferably less than 0.1%.Amino acid racemization in GLP-1 compounds can be detected andquantitated by methods known in the art such as HPLC chromatography(see, for example, Senderoff et al., J. Pharm. Sci. 87:183 (1998)) andthe assay described in Example 6. Thus, suitable pHs for the neutralizedsolution can be readily determined by one of ordinary skill in the art.For most applications, pH values for the neutralized solution can rangefrom between about 6.5 to about 9.0. Preferred pH values are betweenabout 7.0 and about 8.5.

In order to minimize amino acid racemization, it is desirable toneutralize the basic or acidic GLP-1 solution as soon as possible afterdissolution of the insoluble GLP-1 compound. Preferably, the GLP-1solution is neutralized within a sufficiently short period of time sothat there is substantially no racemization or degradation of aminoacids in the GLP-1 compound, e.g., less than 1% of the GLP-1 compoundscontain racemized amino acids and preferably less than 0.1%. As notedabove, the rate of amino acid racemization and other degradationincreases with increasing solution basicity or acidity. Therefore, theamount of time which can lapse between dissolution and neutralizationwithout the substantial formation of degradation or racemizationby-products varies according to the pH of the GLP-1 solution. When thepH of the GLP-1 solution is between about 10.5 and about 12.5 or betweenabout 1.5 and about 2.0, substantially no amino acid racemization ordegradation occurs when the GLP-1 solution is neutralized within aboutthirty minutes after dissolution of the insoluble GLP-1 compound.Preferably, the GLP-1 solution is neutralized within about five minutesafter dissolution. Optionally, the GLP-1 solution can be neutralizedbefore complete dissolution of the insoluble GLP-1 compound, providedthat the undissolved material is removed from the solution beforeisolation of the soluble GLP-1 compound. Undissolved material can beremoved by any suitable means, including by filtration.

Any suitable acid or base can be used to adjust the pH of a solution inthe method of the present invention. Preferably, the acid or base aresuitable for use in the preparation of a pharmaceutical product.Examples of suitable acids include hydrochloric acid, sulfuric acid,acetic acid, oxalic acid and the like. Hydrochloric acid is preferred.Examples of suitable bases include hydroxide bases and carbonate bases.Sodium hydroxide is preferred.

A small amount of solid material, referred to herein as “gelatinousmaterial”, typically remains undissolved in the GLP-1 solution. It ispreferable to remove this material from the GLP-1 solution or theneutralized GLP-1 solution before isolation of the soluble GLP-1compound. In one example, the pH of the GLP-1 solution is adjusted toabout 8.0 before removing the gelatinous material. Removal of gelatinousmaterial can be carried out by any suitable means, including byfiltration.

Isolated soluble GLP-1 compound prepared by the method of the presentinvention can be used as the active ingredient in a pharmaceuticalproduct for the treatment of diseases such as Type II diabetes orobesity. Consequently, it is desirable to remove microbial matter fromthe GLP-1 solution and/or the neutralized solution. Microbial matter istypically removed from solutions with a suitable filter membrane,preferably with a filter membrane having a porosity less than or equalto 0.22 μM. Filtration steps to remove gelatinous material and microbialmaterial can be combined or, alternatively, performed as separate steps.In addition, it is desirable to carry out the method of the presentinvention in a sterile environment consistent with Good ManufacturingPractices when the final GLP-1 compound is being used as apharmaceutical.

Soluble GLP-1 compound can be isolated from the neutralized solution byany suitable method, including by lyophilization, spray drying orcrystallization. Examples of a suitable crystallization methods aredescribed in EP 619,322 by Gelfand at al., and WO 99/30731 by Hoffman,at al. The entire relevant teachings of these references areincorporated herein by reference.

Given the sequence information herein disclosed and the state of the artin solid phase protein synthesis, GLP's can be obtained via chemicalsynthesis. However, it also is possible to obtain some GLP's byenzymatically fragmenting proglucagon using techniques well known to theartisan. Moreover, well known recombinant DNA techniques may be used toexpress GLP's consistent with the invention and are preferred.

The principles of solid phase chemical synthesis of polypeptides arewell known in the art and may be found in general texts in the area suchas Dugas, H. and Penney, C., Bioorganic Chemistry (1981)Springer-Verlag, New York, pgs. 54-92, Merrifield, J. M., Chem. Soc.,85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis,pp. 24-66, Freeman (San Francisco, 1969).

Specific descriptions for the preparation of GLP-1 compounds areprovided in U.S. Pat. Nos. 5,705,483, 5,188,666, 5,512,549, WO 91/11457and WO 98/08871, the entire teachings of which are incorporated hereinby reference.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXAMPLE 1 Preparation of Val⁸-GLP(7-37)OH

Val⁸-GLP-1 (7-37)OH having the amino acid sequenceH₂N-His-Val-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ileu-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-COOH(SEQ ID NO: 5) was prepared using solid phase peptide synthesis methodsand the final product was purified by preparative reversed phasechromatography on a C8 silicon-based resin using 0.1% TFA and anacetonitrile elution gradient. The resultant mainstream was dried bylyophilization. Val⁸-GLP(7-37) can also be prepared recombinantlyaccording to procedures disclosed in U.S. Pat. No. 5,705,483, the entireteachings of which are incorporated herein by reference.

EXAMPLE 2 Conversion of Soluble GLP-1 and GLP-1 Analogs to the InsolubleForm

A. Conversion of Soluble Val⁸-GLP(7-37)OH to the Insoluble Form inAqueous Solution

Val⁸-GLP-1 (7-37)OH (1 gram) lyophilized powder was dissolved in 25 mMammonium bicarbonate, pH 8.0 (100 ml). The solution was stirred with aTEFLON stir bar at about 200 rpm at 21° C. (ambient temperature) for 16hours. At varying times, usually within 30 minutes, the solution becamehazy. On continued stirring, the haze became visibly more dense. Thekinetics of the turbidity were measured by rate of formation ofturbidity, i.e. absorbance at 340 nm. The precipitated protein washarvested either by filtration, e.g. Whatman 1 filter disc, orcentrifugation. The supernatant was assayed for protein content eitherby Biuret Colorimetric Assay obtained from Pierce (Rockford, Ill.) or UVabsorbance at 280 nm. By either method, the protein precipitateconstituted in excess of 90% of the starting material, i.e. there wasless than 10% of the protein remaining in the supernatant fraction. Thecollected precipitate was dried in vacuo, i.e. in a lyophilizer. Theresultant precipitated (0.96 grams) material was soluble in 20 mMphosphate at pH 7.2 at less than 0.01 mg/ml. Solubility was tested bysuspension of the solid in the buffer, adjustment of pH, if necessary,and filtration through a 0.2 micron filter. The protein content of thefiltrate was tested by either Biuret Colorimetric Assay, UV absorbanceat 280 nm, or by high pressure liquid chromatography analysis (HPLC)analysis. The HPLC anaylsis was carried out using a C-18, 5 micron, 300angstrom silica column (Jupiter C-18 column, obtained from Phenomenex,Torrance, Calif.) and an elution gradient with Buffer A (10%Acetonitrile in 0.1% Trifluoroacetic Acid solution) and Buffer B (90%Acetonitrile in 0.1% Trifluoroacetic Acid solution), 25 to 60% Buffer Bover thirty minutes. The column was run at 60° C. at 1.0 ml/min flowrate, detection was at 214 nanometers wavelength and the injectionvolume was 20 microliters.

GLP compounds (GLP-1 (7-37)OH, Val⁸-GLP(7-37)OH andisopropylimidazole⁷-arginine²⁶-GLP-1 (8-37)OH) prepared in this way hada solubility less than 0.1 mg/ml and usually lower than 0.05 mg/ml.Salt, especially sodium chloride, when present at greater than 25 mMgreatly accelerated the rate of formation of the insoluble material.

B. Effect of Stirring on Conversion of Soluble Val⁸-GLP(7-37)OH to theInsoluble Form over Time

Three different lots of Val⁸-GLP-1 (7-37)OH (QIY 17, QIY 18 and 361EM7)were solubilized at 1 mg/ml in 20 mM phosphate, pH 7.4 and the final pHadjusted to 7.4 with 1 N sodium hydroxide. Two to ten milliliter samplesof each solution were transferred to two different sets of glass vials.One set of samples was stirred while the other was allowed to standwithout stirring. The turbidity at varying times was measured bydetermining the absorbance at 340 nm. The results are shown in TheFIGURE. Time courses of stirred solutions are designated with an “s”.

As can be seen from The FIGURE, turbidity of the stirred solutionsincreases far more rapidly in the stirred solutions than in solutionsthat were allowed to stand. Because turbidity is indicative of proteinprecipitation, this result is consistent with the rate of formation ofthe insoluble form being accelerated by increased exposure to air causedby stirring.

C. Conversion of Soluble Val⁸-GLP-1 (7-37)OH to the Insoluble Form byTangential Flow

Val⁸-GLP(7-37)OH was subjected to filtration by tangential flow. Duringfiltration, the retentate solution thickened and became more viscous andVal⁸-GLP(7-37)OH formed a gelatinous material on the filter membranes.The formation of the gelatinous material occurred within 30 minutes ofstarting the filtration; the rate of formation was increased in thepresence of salt. The “insolubilized” protein was collected byfiltration or centrifugation and dried in a vacuum oven or alyophilizer. Precipitation in the presence or absence of salt produced aproduct which was soluble at less than 0.1 mg/ml in a neutral pH buffer.

D. Conversion of Soluble Val⁸-GLP-1 (7-37)OH to the Insoluble Form inAcetic Acid/Acetonitrile

Val⁸-GLP-1 (7-37)OH incubated at between 2° to 6° C. in a matrix of 35mM acetic acid with 25% acetonitrile, apparent pH 3.3. With no stirring,preparations developed gelatinous occlusions over about 1 monthincubation. Agitation increased the rate of gel formation. The gel wascollected by centrifugation and dried in vacuo and displayed asolubility of less than 0.1 mg/ml at neutral pH.

EXAMPLE 3 Secondary Structure of Soluble and Insoluble Val⁸-GLP-1(7-37)OH

Four different batches of purified, lyophilized Val-8 GLP-1 (7-37)OHwere analyzed for secondary structure characteristics by FourierTransform Infra-red Spectroscopy (FTIR) to assess alpha helix and betasheet content of the preparations. Deconvolution was done on the Amide Iregion (1750-1600 cm⁻¹) using BioRad Win-IR Pro Software version 2.5.The results are shown below in the Table 1:

TABLE 1 1657 cm⁻¹ 1624 + 1696 cm⁻¹ Batch Number Alpha helix, % totalbeta sheet, % 1 62.7 31.4 2 62.8 29.3 3 63.6 30.6 4 63.6 28.9 MEAN 30.1Sigma 1.2The data in the Table show that the lyophilized powder has high alphahelix content (over 60%) and a relatively low beta sheet content (30% orless).

EXAMPLE 4 Conversion of Insoluble Val⁸-GLP-1 (7-37)OH to SolubleVal⁸-GLP-1 (7-37)OH by Base Excursion

Five samples of Val⁸-GLP-1 (7-37)OH were prepared as described below:

Sample 1

Lyophilized powder of Val⁸-GLP-1 (7-37)OH was dissolved in 10 mM aceticacid (pH 3) at 1 mg/ml and subjected to isoelectric precipitation by pHadjustment to 5.5 with 1N sodium hydroxide. The resultant precipitatewas collected by centrifugation and dried in vacuo.

Sample 2

An isoelectric precipitate was carried out by approaching pH 5.5 fromthe alkaline side by solubilizing Val⁸-GLP-1 (7-37)OH in 10 mM ammoniumbicarbonate, pH 8.0, then adjusting the pH down with 1 N hydrochloricacid.

Sample 3

A gelled sample was collected by centrifugation from a solution ofVal⁸-GLP-1 (7-37)OH (2.7 mg/ml in 25 mM Acetic Acid, pH 3.3 containing25% acetonitrile) which had been incubated at 4° C. for 3.1 months.

Samples 4 and 5

Val⁸-GLP-1 (7-37)OH was dissolved at 1 mg/ml in 10 mM phosphate (pH 7.2)and stirred until a precipitate formed. The solution was fractionated bycentrifugation to produce a soluble GLP-1 fraction (Sample 4) and aprecipitated fraction (Sample 5). Both fractions were dried in vacuo.

Each of Samples 1-5 was measured for secondary structure characteristicsby Fourier Transform Infrared Spectroscopy (FTIR). In addition, eachSample 1-5 was placed into 20 mM phosphate solution (pH 7.2) and the pHraised to 12.3 by addition of 1N sodium hydroxide for no more than 3minutes. All samples then dissolved and the solutions became clear. ThepH of each solution was then adjusted to 7.0 by addition of 1Nhydrochloric acid. Samples 1A-5A, obtained from Samples 1-5,respectively, were then obtained by lyophilization of the solutions anddried in vacuo. The secondary structural characteristics of Samples1A-5A were assessed by FTIR. The results of the FTIR analysis of Samples1-5 and 1A-5A are shown below in Table 2:

TABLE 2 alpha helix percent beta sheet character percent Sample 1657cm⁻¹ 1624 + 1696 cm⁻¹ 1 24.9 69.8 1A broad peak at 1583 cm⁻¹ interferedwith analysis 2 51.9 40.5 2A 85.9 none observed 3 25.5 70.0 3A 62.2 23.74 75.8 14.7 4A 86.0 none observed 5 12.9 57.3 5A 90.6 none observedAll samples were analyzed by High Performance Liquid Chromatography, asdescribed in Example 2 and shown to contain a single peak whichco-eluted with reference to standard Val⁸-GLP-1 (7-37)OH. This suggeststhat no sample underwent an HPLC discernible chemical change. In eachcase, the base excursion increased the measured alpha helix content ofthe protein and resulted in a product which was soluble at >1 mg/ml in20 mM phosphate buffer, pH 7.2.

In another example of the effects of base excursion on secondarystructure, a purified, lyophilized sample of Val⁸-GLP-1 (7-37)OHpurified by reversed chromatography was either lyophilized (0.1 gram) orprecipitated by addition of sufficient 3.0 M sodium dihydrogen phosphate(pH 3.80) to result in a 0.6 M phosphate solution. The resultantprecipitate was washed with 10 mM sodium acetate at pH 5.5 and thewashed precipitate was dried in vacuo. A second portion of the washedprecipitate was solubilized by raising the pH of the material to 10.5for 30 minutes at room temperature by addition of 1N sodium hydroxide.The pH of that solution was then reduced to 7.0 by addition of 1.0 Nhydrochloric acid and the solution was then lyophilized. The secondarystructure of the three samples was determined by FTIR. The results areshown in Table 3:

TABLE 3 percent alpha percent beta Sample helix content sheet contentLyophilized mainstream 71 16 Phosphate precipitate 57 35 pH 10.5excursion 71 13The results show that basic pH excursion reduces the beta sheet contentand increases the alpha helix content of the sample.

EXAMPLE 5 Soluble Val⁸-GLP-1 (7-37)OH Showed Increased BioavailabilityCompared with Insoluble Val⁸-GLP-1 (7-37)OH

High beta sheet-content Val⁸-GLP-1 (7-37)OH was prepared bysolubilization of the lyophilized powder in 20 mM phosphate buffer, pH7.2 and subjecting the solution to stirring overnight at 21° C. Theresultant precipitate was collected, washed with de-ionized water anddried in vacuo. The lyophilized powder starting material and the dried,precipitate were tested for bioavailability by injection into groups ofthree rats. To prepare the samples, the lyophilized powder startingmaterial (soluble) was formulated into a solution at 2 mg/mL in 15 mMphosphate buffer (pH 7.5). The dried precipitate was then made into aslurry in 10 mM phosphate (pH 7.2); the insoluble material was made intoa suspension at 16.5 mg solid per mL in 15 mM phosphate buffer (pH 7.5).The formulated materials were injected subcutaneously at 80 and 800microgram/kilogram body weight, while the precipitate slurry wasinjected at 10 times that, i.e. 8000 micrograms/kilogram. The maximumconcentration in serum samples taken from the rats was determined byEnzyme Linked Immunoabsorbent Assay to be 11-15 nanogram/ml for theformulated material and 0.4 to 0.6 ng/ml for the slurry. The area underthe curve was about 1% or less for the slurry as compared to theformulated material.

EXAMPLE 6 HPLC analysis of Val⁸-GLP-1 (7-37)OH exposed to high pH forshort periods

Solubilized Val⁸-GLP-1 (7-37)OH was exposed to pH 12.3 for 10 minutes atroom temperature. No discernible change was observed in the HPLCprofile. However, an HPLC analysis, performed as described in Example 2,of solubilized Val⁸-GLP-1 (7-37)OH, exposed to pH 12.3 for severalhours, showed several new peaks which were more polar, i.e., elutedearlier. Three new peaks were prominent in this profile and grew at0.36, 0.06 and 0.64% per hour. A five minute exposure would result in0.09% degradation according to this data. Fractionation of these peaksand analysis by mass spectroscopy (ESI) and Edman degradation analysisshowed the three peaks have identical masses (3384.4+/−0.5 amu) andsequences through the 31 cycles of the peptides. This result isconsistent with the isomerization of amino acid residues from the L tothe D conformation (see, for example, Senderoff, et al., J. Pharm. Sci.87:183 (1998).

This data is consistent with the conclusion that the currently observedconformational effects of base excursion on Val⁸-GLP-1 (7-37)OH, asmeasured by FTIR and solubility, are not due to L to D isomerization,which is manifest in new HPLC peaks.

EXAMPLE 7 Conversion of Insoluble Val⁸-GLP-1 (7-37)OH to SolubleVal⁸-GLP-1 (7-37)OH by Acid Excursion

Insoluble Val⁸-GLP-1 (7-37)OH was suspended in de-ionized water at 2 mgsolid/ml. The pH of one aliquot was dropped to 1.74 by addition of 12microliters of 85% phosphoric acid/ml solution. The pH of a secondaliquot was dropped to pH 1.92 by addition of 5.5 microliters of 10%hydrochloric acid/ml solution. Each sample was gently stirred for 15minutes. The samples were adjusted to pH 7.4 by addition of 10% sodiumhydroxide and then each was filtered through a 0.2 micron filteredmembrane. The soluble portion was lyophilized and analyzed for secondarystructure. Infrared analysis of the products showed that the majorabsorbance in the amide region to be at 1657 cm⁻¹, consistent with theformation of soluble Val⁸-GLP-1 (7-37)OH. HPLC analysis, as described inExample 6, showed no detectable racemization.

Recovered yield was 77.5% for the hydrochloric acid treated sample and6.4% for the phosphoric acid treated sample.

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method of preparing a Glucagon-Like Peptide 1 (GLP-1) compound thatis soluble in aqueous solution at pH 7.4, said GLP-1 compound beingreferred to as a “soluble GLP-1 compound”, from the corresponding GLP-1compound that is insoluble in aqueous solution at pH 7.4, said GLP-1compound being referred to as an “insoluble GLP-1” compound, said methodcomprising the steps of: a) dissolving the insoluble GLP-1 compound inaqueous base to a pH between 9.0 and 12.5 or in aqueous acid to a pHbetween 1.5 and 6.5 to form a GLP-1 solution; b) neutralizing the GLP-1solution to a pH between 6.5 and 9.0 at which no amino acid racemizationof the dissolved GLP-1 compound occurs; and c) isolating soluble GLP-1compound from the neutralized solution of step b) .
 2. The method ofclaim 1 wherein the insoluble GLP-1 compound is GLP-1(7-34),GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide form thereof,having at least one of the following modifications: a) substitution ofglycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine,alanine, valine, isoleucine, leucine, methionine, phenylalanine,arginine, or D-lysine for lysine at position 26 and/or position 34; orsubstitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,phenylalanine, lysine, or a D-arginine for arginine at position
 36. 3.The method of claim 1 wherein the insoluble GLP-1 compound isGLP-1(7-34), GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or the amide formthereof, having at least one of the following modifications: a)substitution of glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,phenylalanine, arginine, or D-lysine for lysine at position 26 and/orposition 34; or substitution of glycine, serine, cysteine, threonine,asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine,methionine, phenylalanine, lysine, or a D-arginine for arginine atposition 36; b) substitution of an oxidation-resistant amino acid fortryptophan at position 31; c) substitution of at least one of: tyrosinefor valine at position 16; lysine for serine at position 18; asparticacid for glutamic acid at position 21; serine for glycine at position22; arginine for glutamine at position 23; arginine for alanine atposition 24; and glutamine for lysine at position 26; d) substitution ofat least one of: glycine, serine, or cysteine for alanine at position 8;aspartic acid, glycine, serine, cysteine, threonine, asparagine,glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine,or phenylalanine for glutamic acid at position 9; serine, cysteine,threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine,leucine, methionine, or phenylalanine for glycine at position 10; andglutamic acid for aspartic acid at position 15; or e) substitution ofglycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine,alanine, valine, isoleucine, leucine, methionine, or phenylalanine, orthe D- or N-acylated or alkylated form of histdine for histidine atposition 7; wherein, in the substitutions in a), b), d), and e), thesubstituted amino acids can optionally be in the D-form and the aminoacids substituted at position 7 can optionally be in the N-acylated orN-aklkylated form.
 4. The method of claim 1 wherein the pH of theaqueous base is between 10.5 and 12.5.
 5. The method of claim 4 whereinthe pH of the aqueous base is between 12.1 and 12.5 and the pH of theneutralized solution of step b) is between 7.0 and 8.5.
 6. The method ofclaim 1 wherein the pH of the aqueous acid is between 1.5 and 2.0. 7.The method of claim 6 wherein the pH of the neutralized solution of stepb) is between 7.0 and 8.5.